A novel R2R3-MYB transcription factor PpMYB5 assisting Ppbbx24-del positively regulates anthocyanin biosynthesis in ‘Red Zaosu’ pear

Abstract

Anthocyanins are vital pigments that play a crucial role in the coloration of various fruits. Our previous study identified a mutant Ppbbx24-del protein in the ‘Red Zaosu’ pear that positively regulates anthocyanin biosynthesis. However, this mutant protein exhibited nucleo-cytoplasmic localization due to the lack of the NLS domain. We hypothesized that a transcription factor in ‘Red Zaosu’ pear interacts with Ppbbx24-del, facilitating its nuclear translocation for regulatory function. In this study, a PpMYB5 was screened by Y2H assay using the Ppbbx24-del as bait, which was an R2R3-MYB transcription factor and significantly up-expressed in ‘Red Zaosu’ compared to ‘Zaosu’. Pull-down, Y2H and BiFC assays confirmed that PpMYB5 could interact with both mutant Ppbbx24-del and common PpBBX24. Notably, co-expression experiments revealed that PpMYB5 facilitated the nuclear translocation of Ppbbx24-del. Transient expression assays in ‘Zaosu’ pear fruits demonstrated that PpMYB5 alone failed to induce anthocyanin accumulation, but its co-expression with Ppbbx24-del significantly enhanced the anthocyanin content of fruit peel compared to Ppbbx24-del alone. This synergistic effect was accompanied by significant upregulation of key anthocyanin biosynthetic genes, including PpCHS and PpCHI. Additionally, dual-luciferase assays demonstrated that PpMYB5 not only enhanced the activation effect on the promoters of PpCHS and PpCHI by Ppbbx24-del but also had the same effect on the promoter of PpMYB5. Our findings indicate that PpMYB5 and Ppbbx24-del form a crucial regulatory module that finely regulates anthocyanin synthesis in pear.

Acknowledgements

We thank the National Natural Science Foundation of China (32072531), China Agriculture Research System-Pear (CARS-28), and Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2016-RIP) for funding this work.

Authors contributions

S.L. executed the experiments and drafted the manuscript. X.L. performed the experiments. F.W., Y.Z., and L.Q. revised the manuscript. C.O. designed and wrote the manuscript. S.J. and H.L. provided supervision, guidance, and support to S.L. and supervised the project and wrote the manuscript.

Data availability

The original contributions presented in the study are included in Supplementary Materials.

Conflicts of interest statement

The authors declare no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

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